Shelly Seward

Ritonavir blocks AKT signaling, activates apoptosis and inhibits migration and invasion in ovarian cancer cells

BackgroundOvarian cancer is the leading cause of mortality from gynecological malignancies, often undetectable in early stages. The difficulty of detecting the disease in its early stages and the propensity of ovarian cancer cells to develop resistance to known chemotherapeutic treatments dramatically decreases the 5-year survival rate. Chemotherapy with paclitaxel after surgery increases median survival only by 2 to 3 years in stage IV disease highlights the need for more effective drugs. The human immunodeficiency virus (HIV) infection is characterized by increased risk of several solid tumors due to its inherent nature of weakening of immune system. Recent observations point to a lower incidence of some cancers in patients treated with protease inhibitor (PI) cocktail treatment known as HAART (Highly Active Anti-Retroviral Therapy).ResultsHere we show that ritonavir, a HIV protease inhibitor effectively induced cell cycle arrest and apoptosis in ovarian cell lines MDH-2774 and SKOV-3 in a dose dependent manner. Over a 3 day period with 20 μM ritonavir resulted in the cell death of over 60% for MDAH-2774 compared with 55% in case of SKOV-3 cell line. Ritonavir caused G1 cell cycle arrest of the ovarian cancer cells, mediated by down modulating levels of RB phosphorylation and depleting the G1 cyclins, cyclin-dependent kinase and increasing their inhibitors as determined by gene profile analysis. Interestingly, the treatment of ritonavir decreased the amount of phosphorylated AKT in a dose-dependent manner. Furthermore, inhibition of AKT by specific siRNA synergistically increased the efficacy of the ritonavir-induced apoptosis. These results indicate that the addition of the AKT inhibitor may increase the therapeutic efficacy of ritonavir.ConclusionOur results demonstrate a potential use of ritonavir for ovarian cancer with additive effects in conjunction with conventional chemotherapeutic regimens. Since ritonavir is clinically approved for human use for HIV, drug repositioning for ovarian cancer could accelerate the process of traditional drug development. This would reduce risks, limit the costs and decrease the time needed to bring the drug from bench to bedside.

Sulforaphane induces cell cycle arrest by protecting RB-E2F-1 complex in epithelial ovarian cancer cells

BackgroundSulforaphane (SFN), an isothiocyanate phytochemical present predominantly in cruciferous vegetables such as brussels sprout and broccoli, is considered a promising chemo-preventive agent against cancer. In-vitro exposure to SFN appears to result in the induction of apoptosis and cell-cycle arrest in a variety of tumor types. However, the molecular mechanisms leading to the inhibition of cell cycle progression by SFN are poorly understood in epithelial ovarian cancer cells (EOC). The aim of this study is to understand the signaling mechanisms through which SFN influences the cell growth and proliferation in EOC.ResultsSFN at concentrations of 5 - 20 μM induced a dose-dependent suppression of growth in cell lines MDAH 2774 and SkOV-3 with an IC50 of ~8 μM after a 3 day exposure. Combination treatment with chemotherapeutic agent, paclitaxel, resulted in additive growth suppression. SFN at ~8 μM decreased growth by 40% and 20% on day 1 in MDAH 2774 and SkOV-3, respectively. Cells treated with cytotoxic concentrations of SFN have reduced cell migration and increased apoptotic cell death via an increase in Bak/Bcl-2 ratio and cleavage of procaspase-9 and poly (ADP-ribose)-polymerase (PARP). Gene expression profile analysis of cell cycle regulated proteins demonstrated increased levels of tumor suppressor retinoblastoma protein (RB) and decreased levels of E2F-1 transcription factor. SFN treatment resulted in G1 cell cycle arrest through down modulation of RB phosphorylation and by protecting the RB-E2F-1 complex.ConclusionsSFN induces growth arrest and apoptosis in EOC cells. Inhibition of retinoblastoma (RB) phosphorylation and reduction in levels of free E2F-1 appear to play an important role in EOC growth arrest.

MicroRNA-101 Inhibits Growth of Epithelial Ovarian Cancer by Relieving Chromatin-Mediated Transcriptional Repression of p21 waf1/cip1

ABSTRACTPurposeMicroRNA-101 (miR-101) expression is negatively associated with tumor growth and proliferation in several solid epithelial cancers. Enhancer of zeste homolog 2 (EzH2) appears to be a functional target of miR-101. We explore the role of miR-101 and its interaction with EzH2 in epithelial ovarian carcinoma (EOC).MethodsIn situ hybridization (ISH) for miR-101 was performed on EOC patient tissues and normal controls. EOC cell lines were transfected with miR-101 and subjected to growth analysis and clonogenic assays. Cell motility was assessed by Boyden chamber and wound-healing assays. P21waf1/cip1 and EzH2 interaction was assessed by Chromatin Immunoprecipitation (ChIP) assay in MDAH-2774 cells. SCID mice were assessed for tumor burden after injection with miR-101 or control vector-treated MDAH-2774 cells.ResultsISH analysis revealed a decrease in miR-101 expression in EOC compared with normal tissue. MiR-101 re-expression in EOC cell lines resulted in increased apoptosis, decreased cellular proliferation, invasiveness, and reduced growth of tumor xenografts. CHIP assays revealed that re-expression of miR-101 inhibited the interaction of EzH2 with p21waf1/cip1 promoter.ConclusionsMiR-101 re-expression appears to have antitumor effects, providing a better understanding of the role of miR-101 in EOC.

Expression of GLUT-1 in epithelial ovarian carcinoma: Correlation with tumor cell proliferation, angiogenesis, survival and ability to predict optimal cytoreduction

OBJECTIVE GLUT-1 is involved at various steps in the processes of tumor progression. The objective of this study was to examine the relationship between GLUT-1 expression and tumor proliferation and angiogenesis in epithelial ovarian carcinoma. MATERIALS AND METHODS Specimens from 213 patients with epithelial ovarian carcinoma were evaluated by immunohistochemistry for GLUT-1, Ki-67, and vascular endothelial growth factor. Tumor microvessel density was assessed with CD34 immunostaining. We investigated the relationships between GLUT-1 expression and clinicopathologic characteristics, tumor angiogenesis (tumor MVD and vascular endothelial growth factor expression), and tumor proliferation (Ki-67). The effect of GLUT-1 expression on patient survival and on the volume of residual disease after cytoreduction was determined. RESULTS There was a significant positive correlation between expression of GLUT-1, Ki-67, and microvessel density. In univariate survival analysis, high GLUT-1 expression, high Ki-67 expression and high tumor microvessel density showed a significant impact on patient survival (p=0.0001). In multivariate analysis including patients with all tumor stages, after controlling for age, race, stage, grade, MVD, and the 3 markers (GLUT-1, Ki-67 and VEGF), only age (HR 1.5; 95% CI 1-2.3), stage (HR 3.6; 95% CI 1.8-7.5) and grade (HR 2.3; 95% CI 1.2-4.5) retained their significance as independent poor prognostic factors. Tumors simultaneously overexpressing GLUT-1 and Ki-67 were less likely to be optimally cytoreduced as compared to tumors overexpressing only one or neither of those two markers (OR: 3.8, p=0.01). CONCLUSION Expression of GLUT-1 correlates with tumor proliferation and microvessel density in epithelial ovarian carcinoma. In addition, patients with rapidly proliferating advanced stage tumors overexpressing GLUT-1 have a lesser chance for optimal cytoreduction.

EZH2 blockade by RNA interference inhibits growth of ovarian cancer by facilitating re-expression of p21waf1/cip1 and by inhibiting mutant p53

The enhancer of zeste homolog 2 (EZH2) methyltransferase is a transcriptional repressor. EZH2 is abnormally elevated in epithelial ovarian cancer (EOC). We demonstrated that EZH2 knockdown inhibited cell growth, activated apoptosis, and enhanced chemosensitivity. Further, silencing of EZH2 resulted in re-expression of p21(waf1/cip1) and down-regulation of mutant p53. Finally, EZH2 knockdown contributed to attenuated EOC growth in SCID mice.

Restoration of E-cadherin expression in pancreatic ductal adenocarcinoma treated with microRNA-101.

PURPOSE To investigate the possibility of inhibiting the progression of pancreatic ductal adenocarcinoma (PDAC) by facilitating the expression of E-cadherin through the enforced expression of microRNA-101 (miR-101). METHODS In situ hybridization was conducted with archival tissue using a double digoxigenin-labeled probe. Chromatin immunoprecipitation (ChIP) assay was conducted with EZ-Magna ChIPTM A. Gene profile analysis, Western blot, and immunoprecipitation assays were performed using standard protocols. RESULTS We found that decreased miR-101 expression observed in archival patient tissues was significantly associated with poor prognosis indicated by low-intensity staining in high-grade tumors. ChIP assays using anti-enhancer of zeste homolog 2 (EZH2) antibodies indicated not only the interaction of EZH2 to the CDH1 (E-cadherin) promoter, but also that this interaction was significantly diminished in cells transfected with pre-miR-101. We observed a global downregulation of trimethylated lysine 27 of H3 histone (H3K27me3) along with upregulation of the enzymes histone deacetylase -1 and -2 with the re-expression of miR-101. Further, we observed lesser levels of transcriptional factors that inhibit the CDH1 promoter with pre-miR-101 treatment. Western blot analysis confirmed the enhanced E-cadherin expression. PANC-1 cells transduced with pre-miR-101 displayed markedly attenuated growth in SCID mice. CONCLUSION These results suggest the potential therapeutic use of miR-101-enforced expression for inhibition of PDAC.

Prognostic impact of laterality in malignant ovarian germ cell tumors.

Objective: To compare the survival of patients with bilateral versus unilateral malignant ovarian germ cell tumors (OGCT). Methods: Patients with a diagnosis of OGCT were identified from the Surveillance, Epidemiology, and End Results Program for the period 1988 to 2006 and were divided into bilateral and unilateral subgroups. Only surgically treated patients were included. Histologic types were grouped into dysgerminoma, malignant teratoma, and mixed germ cell tumors with pure nondysgerminoma cell tumors. Statistical analysis using Wilcoxon rank sum test, Kaplan-Meier survival methods, and Cox proportional hazards regression model were performed. Results: In 1529 patients with OGCT, 1463 (95.7%) were unilateral and 66 (4.3%) were bilateral. Bilaterality was more common with dysgerminomas (6.5%) and mixed germ cell tumors with pure nondysgerminoma cell tumors (6.25%) than with immature teratomas (1.7%), P < 0.001. Most OGCT (67.3%) were stage I. Bilateral OGCT were more likely than unilateral tumors to be associated with advanced-stage disease (FIGO III and IV, 41% vs 20%, P < 0.04). Overall 5-year survival was 93.6% for unilateral OGCT and 80.7% in bilateral OGCT, P < 0.001. In multivariate analysis, bilaterality was not an independent predictor of survival when controlling for age, histology, stage, and surgical staging (hazard ratio, 1.3; 95% confidence interval, 0.7-2.5; P = 0.40). Conclusions: Compared with unilateral tumors, bilateral OGCT are more often associated with advanced-stage disease, high-risk histology, and poor survival. When other prognostic factors are accounted for, bilaterality was not an independent prognostic predictor of survival.

Radiation-associated endometrial cancer.

OBJECTIVE: To compare prognostic variables and survival of radiation-associated endometrial cancers with sporadic second endometrial cancers. METHODS: Patients with primary cancers of pelvic organs (urinary system, colorectal, cervix, vulva, and vagina) were identified from the Surveillance, Epidemiology, and End Results (SEER) database between 1973 and 2005. Among these patients, those who received pelvic radiation and subsequently developed endometrial cancer formed the radiation-associated endometrial cancers cohort (cases), whereas those who did not receive pelvic radiation but subsequently developed endometrial cancer formed the sporadic second endometrial cancers cohort (controls). Comparisons between radiation-associated endometrial cancers and sporadic second endometrial cancers used &khgr;2, t tests, Kaplan-Meier, and cox proportional hazards analysis. RESULTS: In 205 radiation-associated endometrial cancer patients and 1,001 sporadic second endometrial cancer patients, the mean age of diagnosis was 65 years and 68 years, respectively (P<.001). The mean latency between primary pelvic organ cancer and second endometrial cancer was 110 months for the radiation-associated endometrial cancers and 77 months for the sporadic second endometrial cancers (P=.03). The lesions in the radiation-associated endometrial cancers cohort (compared with sporadic second endometrial cancers) were far more likely to be nonendometrioid histology (76.2% compared with 51%, P<.001), poorly differentiated (58% compared with 28%, P<.001), and advanced stage (International Federation of Gynecology and Obstetrics III and intravenously combined) (43% compared with 16%, P<.001). The 5-year survival rate for radiation-associated endometrial cancers and sporadic second endometrial cancers was 27.1% and 57.1%, respectively (P<.001). In multivariable analysis, after controlling for age, race, histology, stage, grade, and treatment, the hazard ratio for death of sporadic second endometrial cancers was 1.4 (95% (CI) 1.2–3.6; P=.002). CONCLUSION: The radiation-associated endometrial cancers carry a grave prognosis because they are more likely to be nonendometrioid, poorly differentiated advanced stage cancers. The longer latency and extensive spread at diagnosis among radiation-associated endometrial cancers may suggest a possible delay in clinical presentation and diagnosis. LEVEL OF EVIDENCE: II

The prognostic impact of the ratio of positive lymph nodes on survival of epithelial ovarian cancer patients.

To study the prognostic significance of ratio of positive to examined lymph nodes (LNR) on survival of patients with node positive epithelial ovarian cancer (NPEOC).

Therapy-related myeloid leukemia after treatment for epithelial ovarian carcinoma: An epidemiological analysis

OBJECTIVE Therapy related acute myeloid leukemia (t-AML) is a potential late complication of cytotoxic therapy, and it is of particular concern in the treatment of patients with epithelial ovarian carcinoma (EOC) exposed to multiple courses of chemotherapy during the course of their disease. This study examines the incidence, characteristics and clinical outcomes of patients who developed secondary myeloid-type leukemia after a diagnosis of EOC. METHODS National Cancer Institutes Surveillance, Epidemiology and End Results database was pooled for diagnosis of secondary myeloid leukemia after an initial diagnosis of EOC. This group of patients was compared to patients with de novo AML, and to EOC patients who did not develop secondary myeloid leukemia. Demographic, cytopathological and survival data were recorded. Cox Proportional Hazards model was used to calculate hazard ratios (HR) for developing secondary leukemia and to determine the statistically significant variables impacting survival. Kaplan-Meier estimates of survival were obtained and comparisons between the groups were performed using log-rank test. RESULTS One hundred and nine myeloid leukemia cases were identified among 63,359 patients with a prior diagnosis of EOC for an overall incidence of 0.17%. The median latency to development of leukemia was 4 years (range 0-27 years). Median survival from the time of secondary leukemia diagnosis was 3 months and significantly worse than the 6 month median survival in patients with de novo AML (p<0.001). Age at leukemia diagnosis greater than 65 and development of secondary vs. de novo leukemia had a statistically worse prognosis on multivariate analysis (HR of 2.69, 95%CI 2.60-2.78 and 1.81, 95%CI 1.49-2.20 respectively). The development of secondary leukemia was more common with EOC diagnosis made prior to the platinum/taxane era (HR 6.70, 95%CI 3.69-12.18). There was no difference in median survival between EOC patients who developed AML and those who did not. CONCLUSION Development of t-AML is a rare but lethal event among EOC patients, and its incidence has decreased significantly since the use of platinum/taxane-based chemotherapy became the standard of care.